The Effect of siRNA-VEGF on the Growth of REC in Retina Pigment Epithelial Cell and Retinal Endothelial Cell Co-culture System
摘要
Purpose:To investigate the effect of small interfering RNA (siRNA)targeting VEGF of retinal pigment epithelium (RPE) cells on the growth activity of human retinal vascular endothelial cells (RECs) under a co-culture system.Methods:By applying the vector.(pGPU6)-based siRNA plasmid gene silence system,we specifically silenced VEGF expression of RPE cells (ARPE-19) through plasmid (pG-PU6-VEGFA-siRNA)transfection.Reverse transcription polymerase chain reaction(RT-PCR) was applied for selecting the most efficient siRNA segment among three pGPU6-VEGF-siRNA groups (siRNA-1,siRNA-2 and siRNA-3).Treated RPE cells were co-cultured with RECs in a co-culture system made up of a 24-well culture plate and transwell inserts assembled inside During 7-day culture period,the growth ca-pacity of RECs were observed and tested in the form of cell counting assay.Three groups were established in this study:RPE cells transfected with pGPU6-VEGF-siRNA and co-cultured with RECs (group A),RPE cells transfected with siR-NA null vector and co-cultured with RECs (group B),and RECs cultured alone (group C).Results:After transfection,VEGF expression levels of RPE cells in three pGPU6-VEGF-siRNA groups (siRNA-1,siR-NA-2 and siRNA-3)evaluated by RT-PCR were 2.56 ±0.45,1.17 ±0.38 and 4.39 ±0.51,respectively (n =10).siRNA-2 was selected as the foremost segment for transfection (P < 0.05,SNK-q test).During the 7-day co-culture period,the influence upon the growth of RECs was observed.Growth curve of RECs under co-culture showed a lower growth rate in group A than in group B (P <0.05,dunnett’s test),but no significant difference between group A and group C was noted (P>0.05,dunnett’s test).RECs in group A proliferated muchfaster during the first four days post-transfection.Conclusion:Delivery of siRNA targeting VEGF plays an efficient role in down-regulating VEGF expression in RPE cells,therefore modulating the growth activity of RECs under a co-culture system in vitro.The application of this technique may provide novel evidence for the prevention and treatment of retinal neovascularization diseases.
